棉铃虫(Helicoverpa armigera)FK506结合蛋白基因的克隆、表达与纯化

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 115-120.

棉铃虫(Helicoverpa armigera)FK506结合蛋白基因的克隆、表达与纯化

刘小宁, 李芬, 赵文博, 朱燕, 赵洁

(新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室，乌鲁木齐 830046)

收稿日期:2013-11-07 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:刘小宁，女，博士，研究方向：农业昆虫与害虫防治；E-mail：liuxn0103@sina.com；李芬同为本文第一作者
基金资助:
国家自然科学基金项目(31260444)，农业部行业项目(200903033)

Cloning，Expression and Purification of FK506 Binding Protein Gene from Helicoverpa armigera

Liu Xiaoning, Li Fen, Zhao Wenbo, Zhu Yan, Zhao Jie

(College of Life Science and Technology，Xinjiang University，Xinjiang Key Laboratory of Biological Resources and Genetic Engineering，Urumqi 830046)

Received:2013-11-07 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 为了深入了解FK506结合蛋白基因的作用和探索调控棉铃虫CYP6B6的表达机制，基于单酵母杂杂交结果采用RT-PCR的方法从棉铃虫的中肠cDNA中扩增得到了FK506结合蛋白基因，将测序正确的目的片段克隆至原核表达载体pET32a中，在大肠杆菌BL21中用异丙基-β-D-硫代半乳糖(IPTG)诱导表达，通过镍柱离子亲和层析纯化目的蛋白。用SDS-PAGE检测目的蛋白的表达和纯化结果，并用Western blotting进行验证。结果表明，克隆得到的目的基因大小为327 bp，编码108个氨基酸残基，预测蛋白质分子量和等电点分别是11.78 kD和7.87。氨基酸序列分析表明该序列具有完整的开放阅读框，且没有信号肽。重组质粒pET32a-FKBP在大肠杆菌BL21中获得表达，主要以可溶性形式存在，经亲和层析柱纯化获得目的蛋白，Western blotting检测发现纯化的目的蛋白大小正确且纯度高。

关键词: 棉铃虫, FK506结合蛋白基因, 单酵母杂交

Abstract: In order to better understand the role of FK506-binding protein gene and its regulatory mechanism in the CYP6B6 expression of Helicoverpa armigera, the FK506-binding protein cDNA sequence from midgut of H. armigera by RT-PCR was cloned on the basis of result of yeast one-hybrid. The fragment digested by double enzymes was linked to a prokaryotic expression vector pET32a to construct the recombinant expression plasmid pET32a-FK506BP, and then converted into competent Escherichia coli BL21 cells. The fusion protein was induced to express by isopropyl-3-D-thiogalactoside(IPTG), and purified by immobilized metal-chelating affinity chromatography(IMAC)using a Ni²⁺ matrix column. SDS-polyaerylamide gel electrophoresis(SDS-PAGE)and Western blotting analysis were used to examine the fusion protein. The results of sequencing and sequence analysis showed that the open reading frame of the FK506 binding protein gene was 327 bp, encoding 108 amino acid residues with the predicted molecular weight and isoelectric point of 11.78 kD and 7.87, respectively. The predicted protein had no signal peptide. The recombinant containing recombinant pET32a-FK506BP expressed a soluble protein after being induced by IPTG. SDS-PAGE and Western blot analysis indicated that the fusion protein, purified using Ni²⁺ affinity chromatography, had the predicted size and higher purity.

Key words: Helicoverpa armigera, FK506-binding protein, Yeast one-hybrid

刘小宁, 李芬, 赵文博, 朱燕, 赵洁. 棉铃虫(Helicoverpa armigera)FK506结合蛋白基因的克隆、表达与纯化[J]. 生物技术通报, 2014, 0(4): 115-120.

Liu Xiaoning, Li Fen, Zhao Wenbo, Zhu Yan, Zhao Jie. Cloning，Expression and Purification of FK506 Binding Protein Gene from Helicoverpa armigera[J]. Biotechnology Bulletin, 2014, 0(4): 115-120.

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